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Userś Manual

Toxoplasma gondii IgM

µ-capture ELISA
RUO

TOXM0460

96 wells

For Research Use Only – Not for Use in Diagnostic Procedures

Demeditec Diagnostics GmbH

Lise-Meitner-Strasse 2
24145 Kiel – Germany
www.demeditec.com

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CONTENTS
1. INTENDED USE 3
2. PRINCIPLE OF THE ASSAY 3
3. MATERIALS 3
4. STABILITY AND STORAGE 3
5. REAGENT PREPARATION 4
6. SAMPLE COLLECTION AND PREPARATION 4
7. ASSAY PROCEDURE 4
8. RESULTS 5
9. SPECIFIC PERFORMANCE CHARACTERISTICS 5
10. LIMITATIONS OF THE PROCEDURE 6
11. PRECAUTIONS AND WARNINGS 6

ABBREVIATIONS 7
SUMMARY OF TEST PROCEDURE 7
SYMBOLS USED WITH DEMEDITEC ASSAYS 8

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1. INTENDED USE
The Toxoplasma gondii IgM µ-capture ELISA is intended for the determination of IgM class antibodies
against Toxoplasma gondii in human serum or plasma (citrate, heparin).
For research use only - Not for use in diagnostic procedures.

2. PRINCIPLE OF THE ASSAY

The immunoenzymatic determination of specific IgM-class antibodies is based on the ELISA (Enzyme-
linked Immunosorbent Assay) µ-capture technique.
Microtiterplates are coated with anti-human IgM-class antibodies to bind the corresponding antibodies
of the sample. After washing the wells to remove all unbound sample material, horseradish peroxidase
(HRP) labelled antigen is added. This antigen-conjugate binds to the captured specific IgM antibodies.
In a second washing step unbound conjugate is removed. The immune complexes are visualized by
adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this
product is proportional to the amount of specific IgM antibodies in the sample. Sulphuric acid is added
to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using
an ELISA Microtiterplate reader.

3. MATERIALS
3.1. Reagents supplied
1. SORB MT Microtiterplate: 12 break-apart 8-well snap-off strips coated with anti-human IgM-class
antibodies; in resealable aluminium foil.
2. SAM DIL Sample Dilution Buffer: 1 bottle containing 100 mL of phosphate buffer (10 mM) for
sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap; ≤ 0.0015% (v/v)
CMIT/ MIT (3:1).
3. STOP SOLN Stop Solution: 1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use;
red cap.
4. WASH SOLN 20x Washing Buffer (20x conc.): 1 bottle containing 50 mL of a 20-fold
concentrated phosphate buffer (0.2 M), pH 7.2 ± 0.2, for washing the wells; white cap.
5. ENZ CONJ Conjugate: 1 bottle containing 15 mL of peroxidase labelled Toxoplasma gondii
antigen; coloured red; ready to use; black cap; ≤ 0.02% (v/v) MIT.
6. SUB TMB TMB Substrate Solution: 1 bottle containing 15 mL 3,3',5,5'-tetramethylbenzidine
(TMB), < 0.1 %; ready to use; yellow cap.
7. CAL C Positive Control: 1 vial containing 2 mL control; coloured yellow; ready to use; red cap;
≤ 0.02% (v/v) MIT.
8. CAL B Cut-off Control: 1 vial containing 3 mL control; coloured yellow; ready to use; green cap;
≤ 0.02% (v/v) MIT.
9. CAL A Negative Control: 1 vial containing 2 mL control; coloured yellow; ready to use; blue cap;
≤ 0.0015% (v/v) CMIT/ MIT (3:1).
For hazard and precautionary statements see 12.1.
For potential hazardous substances please check the safety data sheet.
3.2. Materials supplied
 1 Cover foil
 1 Instruction for use (IFU)
 1 Plate layout
3.3. Materials and Equipment needed
 ELISA Microtiterplate reader, equipped for the measurement of absorbance at 450/620 nm
 Incubator 37 °C
 Manual or automatic equipment for rinsing Microtiterplate
 Pipettes to deliver volumes between 10 and 1000 µL
 Vortex tube mixer
 Distilled water
 Disposable tubes

4. STABILITY AND STORAGE

Store the kit at 2...8 °C. The opened reagents are stable up to the expiry date stated on the label when
stored at 2...8 °C.

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5. REAGENT PREPARATION
It is very important to bring all reagents and samples to room temperature (20…25 °C) and mix them
before starting the test run!
5.1. Microtiterplate
The break-apart snap-off strips are coated with anti-human IgM-class antibodies. Immediately after
removal of the strips, the remaining strips should be resealed in the aluminium foil along with the
desiccant supplied and stored at 2...8 °C.
5.2. Washing Buffer (20x conc.)
Dilute Washing Buffer 1 + 19; e. g. 10 mL Washing Buffer + 190 mL distilled water. The diluted buffer is
stable for 5 days at room temperature (20…25 °C). In case crystals appear in the concentrate, warm up
the solution to 37 °C e.g. in a water bath. Mix well before dilution.
5.3. TMB Substrate Solution
The reagent is ready to use and has to be stored at 2...8 °C, away from the light. The solution should
be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become
contaminated and should be thrown away.

6. SAMPLE COLLECTION AND PREPARATION

Use human serum or plasma (citrate, heparin) samples with this assay. If the assay is performed within
5 days after sample collection, the samples should be kept at 2...8 °C; otherwise they should be
aliquoted and stored deep-frozen (-70…-20 °C). If samples are stored frozen, mix thawed samples well
before testing. Avoid repeated freezing and thawing.
Heat inactivation of samples is not recommended.
6.1. Sample Dilution
Before assaying, all samples should be diluted 1+100 with Sample Dilution Buffer. Dispense 10 µL
sample and 1 mL Sample Dilution Buffer into tubes to obtain a 1+100 dilution and thoroughly mix with a
Vortex.

7. ASSAY PROCEDURE
Please read the instruction for use carefully before performing the assay. Result reliability depends on
strict adherence to the instruction for use as described. The following test procedure is only validated
for manual procedure. If performing the test on ELISA automatic systems we recommend increasing the
washing steps from three up to five and the volume of Washing Buffer from 300 µL to 350 µL to avoid
washing effects. Pay attention to chapter 12. Prior to commencing the assay, the distribution and
identification plan for all samples and standards/controls (duplicates recommended) should be carefully
established on the plate layout supplied in the kit. Select the required number of microtiter strips or wells
and insert them into the holder.
Perform all assay steps in the order given and without any delays.
A clean, disposable tip should be used for dispensing each standard/control and sample.
Adjust the incubator to 37 ± 1 °C.
1. Dispense 100 µL standards/controls and diluted samples into their respective wells. Leave well A1
for the Substrate Blank.
2. Cover wells with the foil supplied in the kit.
3. Incubate for 1 hour ± 5 min at 37 ± 1 °C.
4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash
each well three times with 300 µL of Washing Buffer. Avoid overflows from the reaction wells. The
interval between washing and aspiration should be > 5 sec. At the end carefully remove remaining
fluid by tapping strips on tissue paper prior to the next step!
Note: Washing is important! Insufficient washing results in poor precision and false results.
5. Dispense 100 µL Conjugate into all wells except for the Substrate Blank well A1.
6. Incubate for 1 hour ± 5 min at room temperature (20…25°C). Do not expose to direct sunlight.
7. Repeat step 4.
8. Dispense 100 µL TMB Substrate Solution into all wells.
9. Incubate for exactly 15 min at room temperature (20...25 °C) in the dark. A blue colour occurs
due to an enzymatic reaction.
10. Dispense 100 µL Stop Solution into all wells in the same order and at the same rate as for the TMB
Substrate Solution, thereby a colour change from blue to yellow occurs.
11. Measure the absorbance at 450/620 nm within 30 min after addition of the Stop Solution.

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7.1. Measurement
Adjust the ELISA Microtiterplate reader to zero using the Substrate Blank.
If - due to technical reasons - the ELISA Microtiterplate reader cannot be adjusted to zero using the
Substrate Blank, subtract its absorbance value from all other absorbance values measured in order to
obtain reliable results!
Measure the absorbance of all wells at 450 nm and record the absorbance values for each
standard/control and sample in the plate layout.
Bichromatic measurement using a reference wavelength of 620 nm is recommended.
Where applicable calculate the mean absorbance values of all duplicates.

8. RESULTS
8.1. Run Validation Criteria
In order for an assay run to be considered valid, these Instructions for Use have to be strictly followed
and the following criteria must be met:
 Substrate Blank: Absorbance value < 0.100
 Negative Control: Absorbance value < Cut-off
 Cut-off Control: Absorbance value 0.150 – 1.300
 Positive Control: Absorbance value > Cut-off
If these criteria are not met, the test is not valid and must be repeated.

8.2. Calculation of Results

The Cut-off is the mean absorbance value of the Cut-off Control determinations.
Example: Absorbance value Cut-off Control 0.44 + absorbance value Cut-off control
0.42 = 0.86 / 2 = 0.43
Cut-off = 0.43
8.2.1. Results in Units [U]
Sample (mean) absorbance value x 10 = [Units = U]
Cut-off
Example: 1.591 x 10 = 37 U
0.43
8.3. Interpretation of Results
Cut-off 10 U -
Antibodies against the pathogen are present.
Positive > 11 U
There has been a contact with the antigen (pathogen resp. vaccine).
Antibodies against the pathogen could not be detected clearly.
Equivocal 9 – 11 U It is recommended to repeat the test with a fresh sample in 2 to 4 weeks. If the result
is equivocal again the sample is judged as negative.
The sample contains no antibodies against the pathogen.
Negative <9U
A previous contact with the antigen (pathogen resp. vaccine) is unlikely.

9. SPECIFIC PERFORMANCE CHARACTERISTICS

The results refer to the groups of samples investigated; these are not guaranteed specifications.
For further information about the specific performance characteristics please contact Demeditec
Diagnostics GmbH.
9.1. Precision
Intraassay n Mean (E) CV (%)
#1 24 0.465 4.04
#2 24 1.206 6.58
#3 24 1.685 1.92
Interassay n Mean (U) CV (%)
#1 12 28.16 3.30
#2 12 36.98 8.78
#3 12 1.69 13.54
9.2. Specificity
The specificity is defined as the probability of the assay of scoring negative in the absence of the specific
analyte.
It is 99.32% (95% confidence interval: 96.29% - 99.98%).
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9.3. Sensitivity
The sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific
analyte.
It is 98.21% (95% confidence interval: 90.45% - 99.95%).

9.4. Interferences
Interferences with hemolytic, lipemic or icteric samples are not observed up to a concentration of
10 mg/mL hemoglobin, 5 mg/mL triglycerides and 0.5 mg/mL bilirubin.

9.5. Cross Reactivity

The polyclonal B-cell activation induced by Epstein-Barr virus (EBV) may result in false-positive
Toxoplasma gondii IgM antibody results.

10. LIMITATIONS OF THE PROCEDURE

Bacterial contamination or repeated freeze-thaw cycles of the sample may affect the absorbance values.

11. PRECAUTIONS AND WARNINGS

 The test procedure, the information, the precautions and warnings in the instructions for use have
to be strictly followed. The use of the testkits with analyzers and similar equipment has to be
validated. Any change in design, composition and test procedure as well as for any use in
combination with other products not approved by the manufacturer is not authorized; the user
himself is responsible for such changes. The manufacturer is not liable for false results and incidents
for these reasons. The manufacturer is not liable for any results by visual analysis of the samples.
 Only for research use.
 All materials of human or animal origin should be regarded and handled as potentially infectious.
 All components of human origin used for the production of these reagents have been tested for anti-
HIV antibodies, anti-HCV antibodies and HBsAg and have been found to be non-reactive.
 Do not interchange reagents or Microtiterplates of different production lots.
 No reagents of other manufacturers should be used along with reagents of this test kit.
 Do not use reagents after expiry date stated on the label.
 Use only clean pipette tips, dispensers, and lab ware.
 Do not interchange screw caps of reagent vials to avoid cross-contamination.
 Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
 After first opening and subsequent storage check conjugate and standard/control vials for microbial
contamination prior to further use.
 To avoid cross-contamination and falsely elevated results pipette samples and dispense reagents
without splashing accurately into the wells.
 The ELISA is only designed for qualified personnel following the standards of good laboratory
practice (GLP).
 For further internal quality control each laboratory should additionally use known samples.

11.1. Safety note for reagents containing hazardous substances

Reagents may contain CMIT/MIT (3:1) or MIT (see 4.1 oder refers to 4.1)
Therefore, the following hazard and precautionary statements apply.

Warning H317 May cause an allergic skin reaction.

P261 Avoid breathing spray.
P280 Wear protective gloves/ protective clothing.
P302+P352 IF ON SKIN: Wash with plenty of soap and water.
P333+P313 If skin irritation or rash occurs: Get medical advice/ attention.
P362+P364 Take off contaminated and Wash it before reuse.

Further information can be found in the safety data sheet.

11.2. Disposal Considerations

Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of
this kind of waste is regulated through national and regional laws and regulations. Contact your local
authorities or waste management companies which will give advice on how to dispose hazardous waste.

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ABBREVIATIONS

CMIT 5-chloro-2-methyl-4-isothiazolin-3-one
MIT 2-methyl-2H-isothiazol-3-one

SUMMARY OF TEST PROCEDURE

SCHEME OF THE ASSAY

Toxoplasma gondii IgM µ-capture ELISA

Test Preparation
Prepare reagents and samples as described.
Establish the distribution and identification plan for all samples and standards/controls on the plate
layout supplied in the kit.
Select the required number of microtiter strips or wells and insert them into the holder.

Assay Procedure
Substrate Blank Negative Cut-off Positive Sample
(A1) Control Control Control (diluted 1+100)
Negative Control - 100 µL - - -
Cut-off Control - - 100 µL - -
Positive Control - - - 100 µL -

Sample
- - - - 100 µL
(diluted 1+100)

Cover wells with foil supplied in the kit

Incubate for 1 h at 37 ± 1 °C
Wash each well three times with 300 µL of Washing Buffer
Conjugate - 100 µL 100 µL 100 µL 100 µL
Incubate for 1h at room temperature (20…25 °C)
Do not expose to direct sunlight
Wash each well three times with 300 µL of Washing Buffer
TMB Substrate
100 µL 100 µL 100 µL 100 µL 100 µL
Solution
Incubate for exactly 15 min at room temperature (20…25 °C) in the dark
Stop Solution 100 µL 100 µL 100 µL 100 µL 100 µL
Photometric measurement at 450 nm
(reference wavelength: 620 nm)

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SYMBOLS USED WITH DEMEDITEC ASSAYS

Symbol English Deutsch Francais Espanol Italiano
CE-Konfirmitäts- Conforme aux normes
European Conformity Conformidad europea Conformità europea
kennzeichnung européennes
Consult instructions for Gebrauchsanweisung Consulter les Consulte las Consultare le istruzioni
use beachten instructions d’utilisation Instrucciones per l’uso
In vitro diagnostic Ussage Diagnostic Per uso Diagnostica in
In-vitro-Diagnostikum Diagnóstico in vitro
device in vitro vitro
Nur für Seulement dans le Sólo para uso en
RUO For research use only
Forschungszwecke cadre de recherches investigación
Solo a scopo di ricerca

Catalogue number Katalog-Nr. Référence Número de catálogo No. di Cat.

Lot. No. / Batch code Chargen-Nr. No. de lot Número de lote Lotto no

Contains sufficient for Ausreichend für ”n” Contenu suffisant pour Contenido suficiente Contenuto sufficiente
<n> tests/ Ansätze ”n” tests para <n> ensayos per ”n” saggi
Warnhinweise und Avertissements et Tiene en cuenta
Note warnings and Annoti avvisi e le
Vorsichtsmaßnahmen mesures de précaution advertencias y
precautions precauzioni
beachten font attention precauciones
Temperature de Temperatura de Temperatura di
Storage Temperature Lagerungstemperatur
conservation conservacion conservazione
Mindesthaltbarkeits-
Expiration Date Date limite d’utilisation Fecha de caducidad Data di scadenza
datum
Legal Manufacturer Hersteller Fabricant Fabricante Fabbricante

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